Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection

Background Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNβ), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNβ than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNβ overproduction during optineurin deficiency both in vitro and in vivo. Methods To investigate the mechanism of IFNβ overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. Results IFNβ overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNβ overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNβ overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. Conclusion Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNβ overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.


Abstract:
Background Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNβ), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNβ than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNβ overproduction during optineurin deficiency both in vitro and in vivo.

Methods
To investigate the mechanism of IFNβ overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, lethal viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed. Results IFNβ overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNβ overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNβ overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following lethal viral challenge.

Conclusion
Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNβ overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.   Committee (contact via XXX) for researchers who meet the criteria for access to confidential data.
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To investigate the mechanism of IFN overproduction, viral nucleic acids in infected       Optn-KO mice on the C57BL/6 background were generated as described previously [21]. 181 The mice were bred in-house and used under specific pathogen-free conditions. Eight-to-    were used for qPCR and plaque-forming assays to measure viral titres. Medaka were 216 virally challenged by being maintained in water that contained betanodavirus (2 × 10 5 217 50% tissue culture infective dose/ml) and were monitored for 10 days.   StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) and its software were

IFN overproduction by optineurin-defective cells following
308 viral infection is caused by low clearance of viral nucleic acids 309 We investigated a possible interaction between optineurin and viral infection in IFN KO mice (Fig 1A). They showed no difference in morphology, cell growth, or viability 316 compared with WT MEFs (S1 Fig D-F). The activities of NF-B, AP-1, and the IFNB 317 promoter in Optn-KO MEFs were greater than those in WT MEFs after viral infection 318 (Fig 1B and S1 Fig G-I). The secretion of IFN from Optn-KO MEFs was also greater 319 than that from WT MEFs after viral infection with Cantell or Z strains of SeV ( Fig 1C   320 and S1 Fig I) MEFs was higher compared with that in WT cells at 6 and 24 hours after infection ( Fig   324  1D and S1 Fig J). There was no difference in viral DI genome quantity between Optn-KO 325 and WT MEFs immediately after infection, which indicates that the viral susceptibilities 326 of Optn-KO and WT MEFs were similar ( Fig 1E). Next, to measure the ability of Optn- and steady states (Fig 2A and B). Moreover, conversion of LC3-I to LC3-II in Optn-KO 360 MEFs was less than that in WT MEFs (Fig 2C). Differences between Optn-KO and WT

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MEFs in autophagosome signal intensities and LC3 conversion in a starved state, which 362 is a representative autophagic stimulus, were similar to those in steady and virus-infected 363 states (S2 Fig A and B). These results indicate that the autophagic activity of Optn-KO 364 MEFs is always weaker than that of WT MEFs with or without stimulation, such as by  (Figs 3B and C, S3 Fig A).

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Additionally, IFN production by primary astrocytes isolated from Optn-KO mouse pups 412 was greater after viral infection (Fig 3D, S3 Fig B). These results show that optineurin-   431 We examined IFN production by ALS patient cells that carried the optineurin amino 432 acid substitution mutation, p.Q398* or p.E478G, which we described previously [4].

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IFNB promoter activity and IFN production by ALS patient cells with either optineurin 434 mutant were significantly greater than in healthy donor cells (Fig 4A, S4 Fig A and B).